Method of detecting or monitoring a malignant plasma cell disease

ABSTRACT

A method of detecting or monitoring a malignant plasma cell disease comprising detecting in a sample the ratio between the relative amounts of immunoglobulins having:
         (i) a heavy chain class bound to λ light chains; and   (ii) immunoglobulins having the same heavy chain class but bound to κ light chains. More particularly, in one embodiment the method comprises the steps of measuring the relative amounts of the respective immunoglobulins by using:   (i) at least one antibody, or a fragment thereof, specific for the heavy chain class;   (ii) an antibody, or a fragment thereof, specific for λ light chains; and   (iii) an antibody, or a fragment thereof, specific for κ light chains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/883,003, filed Jul. 25, 2007, which is a U.S.national counterpart application of international application serial No.PCT/GB2006/000267 filed Jan. 26, 2006, which claims priority toGB0501741.3, filed Jan. 27, 2005, the entirety of which is herebyincorporated by reference.

The invention relates to assays and methods for detecting or monitoringmalignant plasma cell disease, and to antibodies, or fragments ofantibodies, which are specific for immunoglobulins, the immunoglobulinscomprising a light chain bound to a heavy chain, the isolated antibodyor fragment being further characterised by having specificity for aheavy chain class (also know as heavy chain class) and at the same timehaving specificity for a light chain type. Compositions and methods ofusing the antibodies, for example in the detection of a malignant plasmacell disease, such as a myeloma, are also provided.

Antibody molecules (also known as immunoglobulins) have a twofoldsymmetry and are composed of two identical heavy chains and twoidentical light chains, each containing variable and constant domains.The variable domains of the heavy and light chains combine to form anantigen-binding site, so that both chains contribute to theantigen-binding specificity of the antibody molecule. The basictetrameric structure of antibodies comprises two heavy chains covalentlylinked by a disulphide bond. Each heavy chain is in turn attached to alight chain, again via a disulphide bond. This produces a substantially“Y”-shaped molecule. This is shown schematically in FIG. 1.

Heavy chains are the larger of the two types of chain found inantibodies, with typical molecular mass of 50,000-77,000 Da, comparedwith the smaller light chain (25,000 Da).

There are five main classes or classes of heavy chain which are γ, α, μ,δ and ε which are the constituents heavy chains for: IgG, IgA, IgM, IgDand IgE respectively. IgG is the major immunoglobulin of normal humanserum, accounting for 70-75% of the total immunoglobulin pool. This isthe major antibody of secondary immune responses. It forms a singletetramer of two heavy chains plus two light chains.

IgM accounts for approximately 10% of the immunoglobulin pool. Themolecules, together with J-chains, form a pentamer of five of the basic4-chain structures. The individual heavy chains have a molecular weightof approximately 65,000 and the whole molecule has a molecular weight ofabout 970,000. IgM is largely confined to the intravascular pool and isthe predominant early antibody.

IgA represents 15-20% of human serum immunoglobulin pool. More than 80%of IgA occurs as a monomer. However, some of the IgA (secretory IgA)exists as a dimeric form.

IgD accounts for less than 1% of the total plasma immunoglobulin.

IgE, although scarce in normal serum, is found on the surface membraneof basophils and mast-cells. It is associated with allergic diseasessuch as asthma and hay-fever.

In addition to the five main classes, there are four subclasses for IgG(IgG1, IgG2, IgG3 and IgG4). Additionally there are two subclasses forIgA (IgA1 and IgA2).

There are two types of light chain: Lambda (λ) and Kappa (κ). There areapproximately twice as many κ as λ molecules produced in humans, butthis is quite different in some mammals. Each chain containsapproximately 220 amino acids in a single polypeptide chain that isfolded into one constant and one variable domain. Plasma cells produceone of the five heavy chain types together with either κ or λ molecules.There is normally approximately 40% excess free light chain productionover heavy chain synthesis. Where the light chain molecules are notbound to heavy chain molecules, they are known as “free light chainmolecules”. The κ light chains are usually found as monomers. The λlight chains tend to form dimers.

There are a number of proliferative diseases associated with antibodyproducing cells. FIG. 2 shows the development of B-cell lineage andassociated diseases. These diseases are known as malignant plasma celldiseases. They are summarised in detail in the book “Serum-free LightChain Analysis” A. R. Bradwell, available from The Binding Site Limited,Birmingham, UK (ISBN: 07044 24541).

In many such proliferative diseases a plasma cell proliferates to form amonoclonal tumour of identical plasma cells. This results in productionof large amounts of identical immunoglobulins and is known as amonoclonal gammopathy.

Diseases such as myeloma and primary systemic amyloidosis (ALamyloidosis) account for approximately 1.5% and 0.3% respectively ofcancer deaths in the United Kingdom. Multiple myeloma is the second-mostcommon form of haematological malignancy after non-Hodgkin lymphoma. InCaucasian populations the incidence is approximately 40 per million peryear. Conventionally, the diagnosis of multiple myeloma is based on thepresence of excess monoclonal plasma cells in the bone marrow,monoclonal immunoglobulins in the serum or urine and related organ ortissue impairment such as hypercalcaemia, renal insufficiency, anaemiaor bone lesions. Normal plasma cell content of the bone marrow is about1%, while in multiple myeloma the content is typically greater than 30%,but may be over 90%.

AL amyloidosis is a protein conformation disorder characterised by theaccumulation of monoclonal free light chain fragments as amyloiddeposits. Typically, these patients present with heart or renal failurebut peripheral nerves and other organs may also be involved.

There are a number of other diseases which can be identified by thepresence of monoclonal immunoglobulins within the blood stream, orindeed urine, of a patient. These include plasmacytoma andextramedullary plasmacytoma, a plasma cell tumour that arises outsidethe bone marrow and can occur in any organ. When present, the monoclonalprotein is typically IgA. Multiple solitary plasmacytomas may occur withor without evidence of multiple myeloma. Waldenstrom'smacroglobulinaemia is a low-grade lymphoproliferative disorder that isassociated with the production of monoclonal IgM. There areapproximately 1,500 new cases per year in the USA and 300 in the UK.Serum IgM quantification is important for both diagnosis and monitoring.B-cell non-Hodgkin lymphomas cause approximately 2.6% of all cancerdeaths in the UK and monoclonal immunoglobulins have been identified inthe serum of about 10-15% of patients using standard electrophoresismethods. Initial reports indicate that monoclonal free light chains canbe detected in the urine of 60-70% of patients. In B-cell chroniclymphocytic leukaemia monoclonal proteins have been identified by freelight chain immunoassay.

Additionally, there are so-called MGUS conditions. These are monoclonalgammopathy of undetermined significance. This term denotes theunexpected presence of a monoclonal intact immunoglobulin in individualswho have no evidence of multiple myeloma, AL amyloidosis, Waldenstrom'smacroglobulinaemia, etc. MGUS may be found in 1% of the population over50 years, 3% over 70 years and up to 10% over 80 years of age. Most ofthese are IgG- or IgM-related, although more rarely IgA-related orbi-clonal. Although most people with MGUS die from unrelated diseases,MGUS may transform into malignant monoclonal gammopathies.

In at least some cases for the diseases highlighted above, the diseasespresent abnormal concentrations of monoclonal immunoglobulins or freelight chains. Where a disease produces the abnormal replication of aplasma cell, this often results in the production of moreimmunoglobulins by that type of cell as that “monoclone” multiplies andappears in the blood.

The identification of monoclonal immunoglobulins, and the heavy andlight chains making up those immunoglobulins may be carried out in anumber of ways. Serum protein electrophoresis (SPE) and immunofixationelectrophoresis (IFE) have been used for a number of years to identifythe presence of monoclonal proteins in the serum. Serum proteinelectrophoresis is the standard method for screening for intactimmunoglobulin multiple myeloma and is based upon scanning gels in whichserum proteins have been separated, fixed and stained. There arelimitations associated with this method, including that some samplesfrom patients with myelomas appear normal by electrophoresis. Thisresults in the possibility of missing patients and misdiagnosis of thedisease. Furthermore, the technique does not readily allow for theaccurate quantitative determination of the various proteins identified,particularly at low concentrations. Serum electrophoresis can be used toidentify the presence of free light chains, but the detection limit isbetween 500 mg/L and 2,000 mg/L, depending upon whether or not themonoclonal protein migrates alongside β proteins. Serum proteinelectrophoresis is negative for free light chains in all patients withnon-secretory myeloma.

Immunofixation electrophoresis uses a precipitating antibody against theimmunoglobulin molecules. Whilst this improves the sensitivity of thetest it cannot be used to quantify monoclonal immunoglobulins because ofthe presence of the precipitating antibody. Immunofixationelectrophoresis is also rather laborious to perform and interpretationmay be difficult. Capillary zone electrophoresis is used in manyclinical laboratories for serum protein separation and is able to detectmost monoclonal immunoglobulins. However, when compared withimmunofixation, capillary zone electrophoresis fails to detectmonoclonal proteins in 5% of samples. These so-called “false negative”results encompass low-concentration monoclonal proteins.

Total κ and λ assays have been produced. However, total κ and total λassays are too insensitive for the detection of monoclonalimmunoglobulin or free light chain. This is due to high backgroundconcentrations of polyclonal bound light chains which interfere withsuch assays.

More recently, the applicants have developed a sensitive assay that candetect the free κ light chains and separately, the free λ light chains.This method uses a polyclonal antibody directed towards either the freeκ or the free λ light chains. This is discussed in detail in the book byA. R. Bradwell. The possibility of raising such antibodies was alsodiscussed as one of a number of different possible specificities, in WO97/17372. This document discloses methods of tolerising an animal toallow it to produce desired antibodies that are more specific than priorart techniques could produce. The free light chain assay uses theantibodies to bind to free λ or free κ light chains. The concentrationof the free light chains is determined by nephelometry or turbidimetry.This involves the addition of the test sample to a solution containingthe appropriate antibody in a reaction vessel or cuvette. A beam oflight is passed through the cuvette and as the antigen-antibody reactionproceeds, the light passing through the cuvette is scatteredincreasingly as insoluble immune complexes are formed. In nephelometry,the light scatter is monitored by measuring the light intensity at anangle away from the incident light, whilst in turbidimetry light scatteris monitored by measuring the decrease in intensity of the incident beamof light. A series of calibrators of known antigen (i.e. free κ or freeλ) concentration are assayed initially to produce a calibration curve ofmeasured light scatter versus antigen concentration.

This form of assay has been found to successfully detect free lightchain concentrations. Furthermore, the sensitivity of the technique isvery high.

Because a monoclonal plasma cell of the type causing e.g. multiplemyeloma will produce only one type of antibody with a λ or a κ lightchain, the relative ratio of λ or κ will change.

If the amount of the free λ light chain and the amount of the free κlight chain are known, it is possible to calculate the ratio between thefree λ and the free κ light chains. An example of the results ofplotting serum λ versus serum κ concentrations for patients withdifferent diseases is shown in FIG. 3. The amount of free λ and free κis skewed away from the normal concentrations because of the monoclonalnature of many of these diseases.

Measuring the κ:λ, ratio for free light chains assists in the diagnosisof the disease. Furthermore, if the disease is treated, for example bychemotherapy or radiotherapy, the technique allows the disease to bemonitored. If the disease is successfully being treated, then theconcentrations of free light chains, which have a relatively short lifespan within the blood, will change and move more towards the normalconcentrations observed for normal sera. Moreover, in malignant plasmacell diseases there is often suppression of production of the oppositelight chain, so the κ:λ, ratio can be more sensitive than individual FLCmeasurements.

Haraldsson A., et al. (Ann Clin. Biochem (1991), 28(5): 461-466)discloses ELISA assays for the determination of kappa and lambda ratioswithin total IgG, IgA and IgM.

Chui S. A., et al. (J. Clin. Immunol. (1991), 11(4): 219-223) disclosesstudying patients with primary IgA nephropathy with an ELISA kit. TheELISA uses monoclonal mouse anti-human IgA1 as a solid phase capture andperoxidase-labeled anti-kappa and anti-lambda antibodies. IgAnephropathy is a kidney disease caused when IgA builds up as deposits inkidneys and appears to run in families.

FIG. 4 indicates that not all such diseases produce free light chains.The use of free light chains as a marker for the diseases is thereforenot 100% successful.

The inventors realized that it is possible to produce antibodies andassays that would be able to distinguish between, for example, IgGλ andIgGκ. They therefore tried to produce antibodies which are specific forimmunoglobulins and which had specificity for both a heavy chain classand a light chain type. They have successfully been able to do this.They have also produced assays that allow the rapid quantitativemeasurement of, e.g. IgGλ and IgGκ ratios to allow the rapididentification and/or follow the progression of monoclonal diseasesassociated with production of a specific heavy chain class, or evenheavy chain subclass, in conjunction with a bound λ or κ chain.

By determining the composition of immunoglobulins using an antibodyspecific to a heavy chain class at the same time as a light chain typeor by using a first antibody against a heavy chain class and a secondantibody to determine the light chain type bound to the heavy chain theinventors have produced a sensitive assay for malignant plasma celldiseases. The assays developed allow more sensitive monitoring of thediseases than, for example, by SPE. The greater sensitivity allows thedetection of the clone, for example, when concentrations of themonoclonal protein have fallen below SPE detection limits. Furthermore,this has the potential to identify some biclonal diseases, which mayhave normal light chain ratios.

Moreover, a further advantage is that this assay should not be affectedby renal function. Free λ and free κ are cleared by filtration throughthe kidneys and their concentration is affected by filtration rate. Dueto its size, intact immunoglobulin is cleared by other mechanisms. Thus,rising levels of free light chain but no change in the amount of theimmunoglobulins detected by the current invention may be used toindicate changes in renal clearance only, especially if the κ:λ ratioshowed no changes.

Assays used include ELISA, nephalometry, turbidimetry and flowcytometry. However, the invention is not limited to such assays.

A first aspect of the invention provides a method of detecting ormonitoring a malignant plasma cell disease comprising detecting in asample the ratio between the relative amounts of immunoglobulins having:

(i) a heavy chain class bound to λ light chains; and

(ii) immunoglobulins having the same heavy chain class but bound to κlight chains.

The method preferably quantitatively measures the amounts of the twoimmunoglobulins in the sample.

Preferably, the sample is obtained from tissue or fluid, such as bloodor serum from blood of an animal, such as a mammal, preferably a human.Additionally, it may be possible to identify such proteins in urine.Preferably the sample is assayed in vitro.

The class detected may be selected from IgA, IgG, IgM, IgD and IgE. Theantibodies may also be subclass specific.

The inventors have found that it is possible to use single antibodies todiscriminate between different heavy chain class/light chain typeimmunoglobulins. Hence, the method of the invention may be determinedusing:

(i) an antibody, or a fragment thereof, having specificity for a heavychain class at the same time as having specificity for a first lightchain type in combination with either:

(ii) an antibody, or a fragment thereof, having specificity for theheavy chain class at the same time as having specificity for the secondlight chain type; or

(iii) an antibody, or fragment thereof, having specificity for the heavychain and a further antibody, or fragment thereof, having specificityfor the second light chain type.

In an alternative aspect of the invention, two different parts of theimmunoglobulin to be detected are bound by the antibodies used in theassay. One antibody binds a part of the heavy chain responsible forheavy chain class determination. The second binds a part of the lightchain responsible for identifying it as a κ or λ chain.

Hence, preferably the ratio is determined using:

(i) at least one antibody, or a fragment thereof, specific for the heavychain class;

(ii) an antibody, or a fragment thereof, specific for λ light chains;and

(iii) an antibody, or a fragment thereof, specific for κ light chains.

The presence of the specific antibodies bound to these immunoglobulinsmay be determined using a labeled second antibody. For example, thebinding antibody may be a sheep antibody. The immunoglobulins detectedmay be human immunoglobulins. Hence the presence of sheep antibodiesbound to the human immunoglobulin may be determined using anti-sheepantibodies, e.g. from rabbit or horse.

Use of such antibodies allows the clones produced by the malignantplasma cell diseases to be characterised, even though they may notproduce different ratios of free light chains. Furthermore, instead ofjust measuring free λ or free κ, this test is more specific because itidentifies the heavy chain class as well. This improves thecharacterisation of the monoclonal plasma cell.

Results produced by the inventors indicate that some tumours which donot produce abnormal free κ to free λ ratios, can be identified becauseof the difference in the ratio of, for example, IgGκ and IgGλ or IgAλand IgA, observed.

Measurement of the heavy chain-light chain specific pair is capable ofbeing automated. Furthermore, the technique is more sensitive and allowsthe quantitative determination of the amount of differentimmunoglobulins. It can be used both to aid diagnosis of a disease andalso to monitor the response of the disease to treatment.

The antibodies used in the assay may be heavy chain subclass specific.For example, anti-IgA (IgA1 and IgA2) and anti-IgG (such as IgG1, IgG2,IgG3 or IgG4) antibodies are made by The Binding Site, Birmingham,United Kingdom. This gives more detailed knowledge of the disease beingdetected.

Polyclonal antibodies are preferably used. This allows an improved assayto be produced to monitor different immunoglobulins of, for example, thesame class. Polyclonal antibodies allow some variability betweendifferent heavy chains of the same class to be detected because they areraised against a number of parts of the heavy chain.

The method of the invention may also be used using one or more of thefollowing methods wherein the binding of the antibodies to theimmunoglobulins in the sample is determined by using a nephelometer, aturbidimeter, flow cytometry, ELISA or fluorescently labeled beads suchas Luminex™ beads. Alternatively, a microarray assay may be producedusing the antibodies.

Preferably the ratio is determined by immunoassay, most preferably viaan immunosorbent assay such as ELISA (Enzyme Linked ImmunosorbentAssay). ELISA-type assays per se are well known in the art. They useantibodies, or fragments of antibodies, to detect blood groups, cellsurface markers, drugs and toxins. In the case of the current invention,this type of assay has been used for the method of the invention.

The inventors have found that it is possible to produce ELISA assays atleast as sensitive as Serum Protein Electrophoresis and, in at leastsome cases, more sensitive than using Immunofixation Electrophoresis(IFE), FREELITE™ (The Binding Site, Birmingham, UK) or obtaining totalheavy chain class concentration as nephelometry.

ELISA uses antibodies to detect specific antigens. One or more of theantibodies used in the assay may be labeled with an enzyme capable ofconverting a substrate into a detectable analyte. Such enzymes includehorseradish peroxidase, alkaline phosphatase and other enzymes known inthe art. Alternatively, other detectable tags or labels may be usedinstead of, or together with, the enzymes. These include radioisotopes,a wide range of coloured and fluorescent labels known in the art,including fluorescein, Alexa fluor, Oregon Green, BODIPY, rhodamine red,Cascade Blue, Marina Blue, Pacific Blue, Cascade Yellow, gold; andconjugates such as biotin (available from, for example, Invitrogen Ltd,United Kingdom). Dye sols, metallic sols or coloured latex may also beused. One or more of these labels may be used in the ELISA assaysaccording to the various inventions described herein, or alternativelyin the other assays, labeled antibodies or kits described herein.

The construction of ELISA-type assays is itself well known in the art.For example, a “binding antibody” specific for the immunoglobulin isimmobilised on a substrate. In this case, the immunoglobulin comprises aheavy chain of a particular class, or subclass, attached to either a λlight chain or a κ light chain. The “binding antibody” may beimmobilised onto the substrate by methods which are well known in theart Immunoglobulins in the sample are bound by the “binding antibody”which binds the immunoglobulin to the substrate via the “bindingantibody”.

Unbound immunoglobulins may be washed away.

In ELISA assays the presence of bound immunoglobulins may be determinedby using a labeled “detecting antibody” specific to a different part ofthe immunoglobulin of interest than the binding antibody.

Preferably, flow cytometry is used to detect the binding of theimmunoglobulins of interest and measure the ratios. This technique iswell known in the art for, e.g. cell sorting. However, it can also beused to detect labeled particles, such as beads, and to measure theirsize. Numerous text books describe flow cytometry, such as PracticalFlow Cytometry, 3rd Ed. (1994), H. Shapiro, Alan R. Liss, New York, andFlow Cytometry, First Principles (2nd Ed.) 2001, A. L. Given, WileyLiss.

One of the binding antibodies, such as the antibody specific for theheavy chain class, is bound to a bead, such as a polystyrene or latexbead. The beads are mixed with the sample and the second detectingantibody, such as antibody specific for λ light chains. The detectingantibody is preferably labeled with a detectable label, which binds theimmunoglobulin to be detected in the sample. This results in a labeledbead when the immunoglobulin to be assayed is present.

Labeled beads may then be detected via flow cytometry. Different labels,such as different fluorescent labels may be used for, for example, theanti-λ and anti-κ antibodies. This allows the amount of each type ofimmunoglobulin bound to be determined simultaneously and allows therapid identification of the κ:λ ratio for a given heavy chain class.

Alternatively, or additionally, different sized beads may be used fordifferent antibodies, for example for different class specificantibodies. Flow cytometry can distinguish between different sized beadsand hence can rapidly determine the amount of each heavy chain class ina sample.

Flow cytometry allows rapid identification of the κ/λ ratios for a givenheavy chain class or subclass. This also reduces the need to doimmunofixation tests.

An alternative method uses the antibodies bound to, for example,fluorescently labeled beads such as commercially available Luminex™beads. Different beads are used with different antibodies. Differentbeads are labeled with different fluorophore mixtures, thus allowing theλ/κ ratio for a particular heavy chain class or subclass to bedetermined by the fluorescent wavelength. Luminex beads are availablefrom Luminex Corporation, Austin, Tex., United States of America.

The monoclonal proteins in a sample may be further characterised bylooking at the amount of free λ or free κ light chains in the sample.This is preferably carried out using antibodies specific for free λ orfree κ light chains, such as those sold under the trade mark FREELITE byThe Binding Site Ltd, Birmingham, UK.

A further aspect of the invention provides an immunosorbent assay kit,such as an ELISA assay kit, for use in a method according to anypreceding claim comprising:

-   -   (i) at least one antibody, or a fragment thereof, specific for        the heavy chain class;    -   (ii) an antibody, or a fragment thereof, specific for λ light        chains; and    -   (iii) an antibody, or a fragment thereof, specific for κ light        chains.

The antibodies, labels, etc. are preferably as described above.

Preferably the antibody specific for the heavy chain class isimmobilised to a substrate. The substrate may be a bead, but preferablyis a microtitre plate well.

One or more of the antibodies preferably comprises a detectable label.One or more controls, such as a known amount of a predeterminedmonoclonal protein, such as IgAλ or IgAκ, or a fragment thereof, may beprovided in this and indeed other ELISA, flow cytometry, Luminex,microarrays or other assays described herein. The fragments, when usedwill retain, e.g. antigenic determinants for detecting class or lightchain type.

Flow cytometry kits and Luminex beads are also provided comprising:

-   -   (i) at least one antibody, or a fragment thereof, specific for        the heavy chain class;    -   (ii) an antibody, or a fragment thereof, specific for λ light        chains; and    -   (iii) an antibody, or a fragment thereof, specific for κ light        chains.

The arrangement of the antibodies, labels, etc. are preferably asdescribed above.

Preferably the antibody specific for the heavy chain class isimmobilised onto a substrate, such as a bead, and each type of lightchain specific antibody (ii and iii) is labeled with a differentdetectable label.

Preferably the kit comprises a plurality of different antibodies, orfragments thereof, specific for different heavy chain classes, and eachof the types of different heavy chain class antibodies is attached to adifferent size of bead.

Accordingly a further aspect of the invention provides isolatedantibodies or fragments thereof which are specific for an immunoglobulinheavy chain-light chain pair, said isolated antibody or fragment thereoffurther characterised by having specificity for a heavy chain class andat the same time by having specificity for a light chain type.

Preferably, the antibody is a polyclonal antibody which is capable ofbinding to a heavy chain bound to a light chain, for example thetetramer containing two heavy chains and two light chains. Havingspecificity for a heavy chain class and specificity for a light chainclass is intended to mean that the antibody is able to distinguishbetween different heavy chain classes and also is able to distinguishbetween heavy chains of the same class but which are bound to κ or λlight chains. For example, the antibody is capable of distinguishingbetween IgGλ and IgGκ and is capable of distinguishing between IgGλ andIgAλ. Preferably, the antibody is specific for IgGλ, IgGκ, IgAλ, IgAκ,IgMλ, IgMκ, IgDλ, IgDκ, IgEλ or IgEκ.

The antibody may also be specific for a heavy chain sub-class lightchain combination of the class. For example, it may be specific forIgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. That is, it is capable ofdistinguishing between IgG1κ and IgG2κ.

The fragments of the antibody are capable of specifically binding anddetecting the heavy chain class and/or light chain type and may be a Fabor F(ab′)₂ fragments.

Indeed, the fragments of antibody used in the other aspects of theinvention may also be Fab or F(ab′)₂ fragments.

Preferably, the antibody or fragment is a polyclonal antibody.Polyclonal antibodies allow a plurality of different antibodies to beraised against different epitopes for the specific heavy chain-lightchain combination. This allows for the slight variations betweendifferent immunoglobulins, but which nevertheless comprise the sameheavy chain-light chain combination.

The polyclonal antibodies used in the various aspects of the inventionmay be capable of being produced by the method shown in WO 97/17372.This allows the production of highly specific polyclonal antibodies.

The antibodies or fragments may be immobilised onto a substrate bytechniques well-known in the art. The substrate may, for example, be amicro array or a microtiter plate. Alternatively, the substrate may be apolystyrene and/or latex bead. This allows the antibodies to be used ina number of different assays which are well-known in the art, forexample as shown in EP 0291194 or ELISA assays. The antibody may also belabeled, for example, with a label described above for ELISA. This canbe any entity, the presence of which can be readily detected. The labelmay be a visible label, that is an entity which, in its natural state,is readily visible either to the naked eye or with the aid of an opticalfilter and/or applied stimulation, such as UV light to producefluorescence. For example, minute coloured particles, such as dye sols,metallic sols (e.g. gold) or coloured latex particles, may be used.

Indirect labels, such as enzymes (e.g. alkaline phosphatase andhorseradish peroxidase) can be used, as can radioactive labels such as³⁵S.

Assays using antibodies are well-known in the art.

The antibodies may be used to produce flow cytometry or Luminex™ kits.These techniques are described herein. Preferably such kits comprise theantibody bound to a bead, such as polystyrene. Preferably the kitadditionally comprises a labeled antibody for detecting the presence ofimmunoglobulins from a sample bound, via the antibody of the invention,to the bead.

Preferably the kit comprises two different types of antibodies specificfor different heavy chain classes and/or different light chain types,and the different types of antibodies are supported on different sizesof beads and/or labeled with different detectable labels.

Kits for detection of specific immunoglobulin molecules comprisingantibodies or fragments according to the invention are also provided.

The kits of the invention may additionally comprise antibodies specificfor free λ or free κ light chains.

The kits may additionally comprise one or more of: instructions forusing the kit, substrate, a buffer, label, a preservative or a control.

Preferably, the immunosorbent assays, such as ELISA assays according tothe various aspects of the invention comprise an antibody specific for λlight chains and a second specific for κ light chains, plus an antibodyspecific for the heavy chain class. Alternatively the ELISA comprisesantibodies specific for the same heavy chain class, but different lightchain types. Such assays may use, for example, a captive layer specificfor the heavy chain class, such as IgG or IgA, plus detection antibodies(anti-λ and anti-κ). Alternatively, anti light-chain (e.g. anti-κ) maybe used as a captive layer with a class specific antibody (e.g.anti-IgA).

The invention further provides a method of carrying out a specificbinding assay, preferably in vitro, comprising:

-   -   (i) providing a sample containing immunoglobulin molecules;    -   (ii) contacting the sample with an antibody or a fragment        thereof according to the invention; and    -   (iii) detecting the specific binding of the antibody to an        immunoglobulin molecule.

Preferably, the specific binding step (iii) is detected using anephelometer or a turbidometer. As already indicated, such techniquesare well-known in the art.

Alternatively, the specific binding assay may use an enzyme-linkedimmunosorbent assay (ELISA) to detect step (iii). Colorimetric methodsof the detection of analytes to specific antibodies are known in theart. For example, EP 0291194 discloses immunoassays using test strips todetect various analytes. The document shows the production of suchassays and the methods of detecting the analyte when bound to specificantibodies. Other techniques for producing assay devices are known inthe art.

Preferably, the antibody or fragment thereof is immobilised on a solidsupport by techniques well-known in the art. The method may additionallyprovide the step of providing a labeling reagent capable of non-specificbinding to the immunoglobulin molecule to be assayed and detecting thepresence of the labeled immunoglobulin bound to the antibody orfragment. The labeling reagent may itself be another antibody directedagainst a different part of the immunoglobulin molecule, the separateantibody being labeled with a label, for example, of the sort discussedabove. The presence of the label allows the production of, for example,a sandwich assay, and the identification of binding of the labeledimmunoglobulin to the molecule to be assayed and its binding to thespecific antibody according to the invention.

The methods of the invention further include ways of detecting thepresence of a first immunoglobulin molecule having a specific class andhaving a specific light chain type comprising the use of an antibody ora fragment thereof according to the invention or a method according tothe invention.

The amount of the immunoglobulin molecule may be quantitativelymeasured.

The methods may also further detect and quantify the presence of asecond immunoglobulin molecule having the same specific heavy chainclass as the first immunoglobulin molecule, but a different type oflight chain is measured, for example using a different antibodyaccording to the invention. This allows the ratio between the amounts ofthe first immunoglobulin molecule and the second immunoglobulin moleculeto be determined, for example to identify the ratio between IgGλ andIgGκ. This allows the progression of a disease to be followed oralternatively the treatment of a disease to be followed.

The invention further comprises a method of diagnosing a malignantplasma cell disease in a patient comprising the use of an antibody or afragment thereof according to the invention, or a method according tothe invention. Preferably blood, urine or serum is assayed.

Preferably, the malignant plasma cell disease is selected from: multiplemyeloma, AL amyloidosis, solitary plasmacytoma, extramedullaryplasmacytoma, multiple solitary plasmacytomas, plasma cell leukaemia,Waldenstrom's macroglobulinaemia, B-cell non-Hodgkin lymphomas, B-cellchronic lymphocytic leukaemia or MGUS.

The invention will now be described by way of example only, withreference to the following figures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of an antibody.

FIG. 2 shows the development of B-cell lineage and associated diseases.MGUS means monoclonal gammopathy of undetermined significance.

FIG. 3 shows a κ and λ graph of serum free light chains showing samplesthat would be misidentified as negative using serum proteinelectrophoresis (SPE) and immunofixation electrophoresis (IFE); LCMMmeans light chain multiple myeloma; NSMM means non-secretory multiplemyeloma; IIMM means intact immunoglobulin multiple myeloma. (Drayson M,Tang L X, Drew R, Mead G P, Carr-Smith H, Bradwell A R. Serum freelight-chain measurements for identifying and monitoring patients withNonsecretory multiple myeloma. Blood 2001; 97: 2900-2902; Bradwell A R,Carr-Smith H D, Mead G P, Harvey T C, Drayson M T. Serum test assessmentof patients with Bence Jones myeloma. Lancet 2003; 361: 489-491; Mead GP, Carr-Smith H D, Drayson M T, Morgan G J, Child J A, Bradwell A R.Serum free light chains for monitoring multiple myeloma. Brit. J.Haematol. 2004; 126: 348-354; Lachmann H J, Gallimore R, Gillmore J D,Carr-Smith H D, Bradwell A R, Pepys M B, Hawkins P N. Outcome insystemic AL amyloidosis in relation to changes in concentration ofcirculating free immunoglobulin light chains following chemotherapy.Brit. J. Haematol. 2003; 1223: 78-84.)

FIG. 4 shows the frequency of abnormal serum free light chainconcentrations in patients with different types of multiple myeloma andWaldenstrom's macroglobulinaemia (WM). LCMM means light chain multiplelight chain myeloma; NSMM means non-secretory multiple myeloma. (DraysonM, Tang L X, Drew R, Mead G P, Carr-Smith H, Bradwell A R. Serum freelight-chain measurements for identifying and monitoring patients withNonsecretory multiple myeloma. Blood 2001; 97: 2900-2902; Bradwell A R,Carr-Smith H D, Mead G P, Harvey T C, Drayson M T. Serum test assessmentof patients with Bence Jones myeloma. Lancet 2003; 361: 489-491; Mead GP, Carr-Smith H D, Drayson M T, Morgan G J, Child J A, Bradwell A R.Serum free light chains for monitoring multiple myeloma. Brit. J.Haematol. 2004; 126: 348-354.)

FIG. 5 shows standard curves for (a) IgA₁κ and (b) IgA₁λ for ELISAassays produced by the method described below:

FIGS. 6 to 15 show IgA κ/λ ratio measured by ELISA, as described below(-▴-); κ/λ ratios as determined looking at the free light chains in thesample, not bound to heavy chains (-♦-), and the total IgA in the sample(-•-). SPE scores (Serum Protein Electrophoresis) for each patient foreach day on which samples were taken are also presented.

FIG. 6 Patient No. 7148 FIG. 7 Patient No. 7273 FIG. 8 Patient No. 7283FIG. 9 Patient No. 7255 FIG. 10 Patient No. 7401 FIG. 11 Patient No.70236 FIG. 12 Patient No. 70338 FIG. 13 Patient No. 70382 FIG. 14Patient No. 70392 FIG. 15 Patient No. 70052

FIG. 16 shows the immunoelectrophoresis (IEP) of anti-IgGκ (A). Thephotograph indicates that the antibody reacts well with IgGκ in serumand pure IgGκ. It is also negative against IgGλ and IgMκ and IgAκ. Allcontrol antiserum reacted positively with target proteins.

DEVELOPMENT AND ANALYSIS OF AN IGA κ/λ ELISA ASSAY SYSTEM FOR THEMONITORING OF MULTIPLE MYELOMA Introduction

Multiple myeloma (MM) is a malignant plasma cell disorder accounting forapproximately 10% of haematological malignancies. The disease ischaracterised by clonal proliferation of plasma cells that produce amonoclonal intact immunoglobulin and/or free light chains (FLC). Themonoclonal immunoglobulin is observed in the serum and/or urine of allpatients except 1-2% with non-secretory myeloma. Some patients exhibitan increased frequency of monoclonal free light chains. In addition tobeing used to aid identification of monoclonal gammopathies such as MM,the monoclonal immunoglobulin can be detected and used to monitor thedisease. Various methods are currently used to identify and characterisemonoclonal immunoglobulins. Serum protein electrophoresis (SPE) andimmunofixation electrophoresis (IFE) are two such methods utilised. SPEallows quantitative analysis of monoclonal immunoglobulins, whereas IFEis a qualitative method. More recently FREELITE™ has been developed thatallows nephelometric analysis of free light chains. This assay systemallows rapid testing of samples in comparison to SPE and IFE and inaddition is quantitative, allowing FLC ratios to be calculated. Atpresent no comparable assay system exists to investigate monoclonallight chains attached to heavy chains in MM. This report describes apreliminary assay developed to assess the ability to detect abnormalratios of monoclonal light chains attached to heavy chains. The assaydescribed is an ELISA assay system for the detection of IgAκ and IgAλ,allowing the quantitation of IgAκ/λ ratios.

FREELITE™ is a trademark of The Binding Site (TBS) Ltd, UK.

Methods

Coating of 96 Well Plates with Capture Antibody

Sheep anti-human IgA (TBS product code AU010, affinity purified) wasdiluted to 5 μg/ml in 1×PBS (pH 7.2) Microtitre plates (High bind,Greiner Bio-one) were coated by the addition of 100 μl of the dilutedantiserum to each well. The plates were placed at 4° C. in a humidifiedatmosphere for 18 hours. The contents of the wells were removed and 110μl/well of 50% [v/v] Stabilcoat (biomolecular stabiliser/blocking agent)added for 30 minutes to block non-coated regions of the wells. Followingremoval of the blocking solution, the plates were placed in a vacuumdrier for 1 hour. The plates were sealed in foil bags containingdesiccant and stored at 4° C.

Determination of the Conjugate Dilution

Sheep anti-human kappa-horseradish peroxidase (HRP), affinity purifiedor sheep anti-human lambda-HRP, affinity purified were diluted tovarious dilutions in conjugate diluent (130 nM NaCl, 10% [v/v] HRPconjugate stabiliser, 0.045% [v/v] Proclin 300 (preservative)). Twosheep anti-human IgA coated plates were incubated with IgA controls ofknown concentration (RID IgA NL control 3.963 mg/ml, RID IgA ML control0.05 mg/ml and RID IgA UL control 0.18 mg/ml,) diluted 1/50 in SampleDiluent (1×PBS plus 2% [v/v] Stabilguard (biomolecularstabiliser/blocking agent), 1% [w/v] bovine serum albumin (BSA), 0.05%[v/v] Tween-20, 0.02% [v/v] Kathon (biocide). pH 7.2) for 30 minutes.The plates were washed 3 times with Wash buffer (16×PBS plus 1% [v/v]Tween-20, 0.02% [v/v] Kathon. pH 7.2). One plate was incubated with 100μl/well of the various dilutions of anti-human kappa-HRP and the secondplate with 100 μl/well of anti-human lambda-HRP conjugate of varyingdilutions for 30 minutes. The plates were again washed 3 times with Washbuffer and incubated with 100 μl/well of 3,3′,5,5′ tetramethylbenzidine(TMB) substrate for 30 minutes. The reaction was terminated by theaddition of 100 μl/well of 3 M phosphoric acid. Absorbances weremeasured at 450 nm using a Biotek ELISA plate reader. A conjugatedilution of 1/8000 was chosen for future assays due to it giving thegreatest absorbance range for the three IgA assay controls.

Standard Curves of IgAκ and IgAλ

IgAκ and IgAλ sera were used to assess the sensitivity of the IgA ELISAassay in determining IgAκ and IgAλ concentrations. Purified human IgAκ(5.14 mg/ml) and human IgAλ (1.85 mg/ml) sera were serial diluted usingtripling dilutions to allow the determination of the detectableconcentration range. The ELISA assay method used was as described above.Briefly, IgAκ or IgAλ sera of various dilutions, or IgA controls (asdescribed above) were added to wells at 100 μl/well in duplicate. Afterwashing, either anti-human kappa-HRP (for IgAκ serum plate) oranti-human lambda-HRP (for IgAλ serum plate) conjugate was added, platesincubated and washed, followed by the addition of TMB substrate and 3 Mphosphoric acid.

Effect of Competition on the Standard Curves of IgAκ and IgAλ

To assess the effect of the presence of IgAλ on the IgAκ assay and viceversa, serial dilution of IgAλ or IgAκ serum was carried out across ananti-IgA coated plate, whilst IgAκ or IgA₁λ serum respectively wereserially diluted down the plate. The ELISA method was carried out asdescribed above.

Determination of κ/λ Ratios of Serum Samples from Healthy Adults

IgAκ and IgAλ values were separately determined, from which κ/λ ratioswere calculated. Two sheep anti-human IgA coated plates were incubatedwith 100 μl/well of IgA controls mentioned previously (diluted 1/800 inSample Diluent), IgAκ or IgAλ sera of various dilutions, and serumsamples from healthy adults diluted 1/4000 in sample diluent for 30minutes. The plates were washed 3 times with Wash buffer and incubatedwith 100 μl/well of either anti-human kappa-HRP or anti-human lambda-HRPconjugate at 1/8000 for 30 minutes. The plates were washed 3 times withWash buffer and incubated with 100 μl/well TMB substrate for 30 minutes.The reaction was terminated by the addition of 100 μl/well of 3 Mphosphoric acid. Absorbances were measured at 450 nm using a BiotekELISA plate reader. Results were analysed using KC4 software.

Determination of κ/λ Ratios of Consecutive Myeloma Patient Serum Samples

The IgAκ and IgAλ assays were carried out as described above, with theaddition of myeloma patient serum samples diluted 1/4000 in samplediluent.

Results and Discussion Standard Curves of IgAκ and IgAλ

The linear detection range of the assays were determined as 0.22-2.2μg/ml for IgAκ (FIGS. 5 a) and 0.22-3.2 μg/ml for IgAλ (FIG. 5 b). Toconfirm the concentration range of the standard curves, serial dilutionsof the RID IgA NL control were also carried out and assayed. Thedetectable assay concentration range agreed with those obtained for IgAκand IgAλ (data not shown). It was concluded that serum samples to betested would require a 1/4000 dilution to fall within the linear rangeof the standardised IgAκ and IgAλ curves.

Effect of Competition on the Standard Curves of IgAκ and IgAλ

Studying the effects of competition of IgAλ on the IgAκ assay and viceversa showed IgAλ caused interference to the IgAκ standard curve(leading to a reduction in absorbance) at IgAλ concentrations of 1.6μg/ml and above. IgAκ caused interference to the IgAλ standard curve at5.35 μg/ml and above. These findings indicate that the system requiresfurther optimization but all the patient testing reported here wasperformed at appropriate dilutions to ensure no interference due tocompetition occurred.

Determination of κ/λ Ratios of Serum Samples from Healthy Adults

18 serum samples from healthy adults, obtained from the BloodTransfusion Services, were assayed in duplicate to determine the normalIgA κ/λ ratios for the ELISA assay. The results indicated a normal rangeof 0.6 to 1.2. These values were similar to values in the literaturestating IgA κ/λ ratios of 0.8 to 1.5 by ELISA (Haraldsson et al., 1991)and 1.1 to 1.8 by nephelometry (Chui et al., 1991).

Determination of κ/λ Ratios of Consecutive Myeloma Patient Serum Samples

99 myeloma patient serum samples were obtained from Dept. ClinicalImmunology, University of Birmingham. These serum samples consisted of10 patient samples sets, following their disease state frompresentation, though treatment, and in some cases, into relapse. Formost samples, SPE results, FLC κ/λ ratios via FREELITE™ (normal range0.26-1.65) and total IgA κ/λ values via nephelometry (normal range0.7-3.6 g/l) had previously been obtained. IgA κ/λ ratios were obtainedin the present study via IgAκ and IgAλ ELISA assays, as described in themethods section. The results obtained, and comparisons to the othermethods used to monitor the disease, are discussed below for eachpatient. In some cases, if discrepancies were observed for resultsbetween methods, IFE (Sebia) gels were also produced for disease stateclarification.

Patient Number 7148

IgA κ/λ ratios produced are in agreement with the trend line observedfor FLC κ/λ ratios (FIG. 6). Most of the total IgA values also correlateexcept for the sample obtained 36 days after presentation, for which thetotal IgA value was within the normal range, but IgA and FLC κ/λ ratioswere elevated, with a SPE score of +/−. IFE of this sample indicated thepresence of an IgAκ band. The results for this patient indicate the IgAκ/λ ELISA assays may have similar sensitivity to FREELITE™ and IFE, andmay have increased sensitivity with respect to SPE and total IgA valuesobtained via nephelometry.

Patient Number 7273

IgA κ/λ ratios calculated agree with trend lines observed for FLC Karatios and total IgA values (FIG. 7). SPE scores correspond to mostvalues obtained by the other methods except for the sample obtained 501days after presentation. For this sample the SPE score is +/− but valuesare within normal ranges for all other methods. IFE of this sampleindicates no monoclonal bands. These results show IgA κ/λ ELISA assaysmay be useful when obtaining borderline SPE scores.

Patient Number 7283

The IgA κ/λ ratios trend line obtained for samples of this patientcorresponds to that seen for FLC κ/λ ratios and agree with most of theSPE scores (FIG. 8). The total IgA value is suppressed for the sampleobtained 23 days after presentation, although all other results areelevated. For the sample obtained 49 days after presentation, total IgAis suppressed, the SPE score is negative, yet the IgA κ/λ ratio isabnormal. (The FLC κ/λ ratio is not known for this sample). IFE of thissample indicated the presence of an IgAκ band, showing the IgA κ/λ ratiohas similar sensitivity to IFE.

Patient Number 7255

IgA κ/λ ratios agree with most, but not all, of the data obtained usingthe other detection methods (FIG. 9). No SPE scores have been recordedfor these samples. IgA κ/λ ratios are above the normal range for samplesobtained 1493, 1576 and 2255 days after presentation. However, total IgAvalues are within normal range for the first two of these samples andFLC κ/λ ratios are within normal range for all three samples. IFEconfirms the presence of a monoclonal IgAκ band in all three samples.Therefore the IgA κ/λ ELISA assays may show greater sensitivity thanFREELITE™ for a number of samples.

Patient Number 7401

The trend line observed for IgA κ/λ ratios agrees with previouslyobtained results for FLC κ/λ ratios and total IgA values (FIG. 10). SPEscores were however negative for all samples. IFE confirmed the presenceof an IgAλ band upon initial presentation.

Patient Number 70236

IgA κ/λ ratio and FLC κ/λ ratio trend lines are similar (FIG. 11).However, there are two samples (165 and 263 days after presentation) forwhich the FLC κ/λ ratios and IgA values are within normal range, SPEscores are negative, but the IgA κ/λ ratios are abnormal. IFE confirmsthe presence of an IgAλ band. These results suggest in some cases theIgA κ/λ ELISA assays are more sensitive than FREELITE™, total IgA valuesobtained via nephelometry and SPE, and of equal sensitivity to IFE.

Patient Number 70338

All methods used to investigate the amount of monoclonal immunoglobulinpresent in the samples gave similar results. The IgAκ/λ ratios producedin the current study agreed with these results (FIG. 12).

Patient Number 70382

All results correlate with each other. Of note is the FLC ratio uponpresentation. This is only just outside the normal range at 0.24,whereas all other methods, including the IgA κ/λ ELISA assays, indicatemuch greater abnormal levels (FIG. 13).

Patient Number 70392

IgA κ/λ ratio and FLC Ka ratio trend lines are similar (FIG. 14).However, FLC κ/λ ratios are only just above normal levels uponpresentation, whereas IgA κ/λ ratios are highly elevated and agree withall other results.

Patient Number 70052

IgA κ/λ ratios are abnormal and agree with values for total IgA.However, SPE scores are unclear, and suggest negative results for allsamples. Furthermore, all FLC κ/λ ratios are within the normal range(FIG. 15). IFE for the first four samples confirms the presence of anIgAλ band in these samples. These results suggest this patient may notsecrete free light chains and therefore all results are negative withthe FREELITE™ assay. They also suggest the IgA κ/λ ELISA assays would bea useful alternative when such a scenario occurs.

In conclusion, for the myeloma patient samples tested, the IgA κ/λ ELISAassays have been shown to be as sensitive as IFE, and in some cases moresensitive than using SPE, FREELITE™ and obtaining total IgA values vianephelometry. There are no incidences in which the IgA κ/λ ELISA assayshave been shown to be less sensitive than the other methods. As themajority of the results obtained using the current ELISA based assaysystem agree with those of the FREELITE™ assay system, it suggests bothmethods are correctly measuring κ/λ ratios to allow investigation ofmonoclonal immunoglobulins characteristic of multiple myeloma.

As the field of immuno-diagnostics continues to develop, many may beconsidering the potential of developing multiplex assay systems thatallow the simultaneous characterisation of a large number of analytes.If the use of multiplexing systems becomes more feasible within theclinical environment in future years, it may be possible to adapt theIgA κ/λ ELISA assays described here to allow simultaneous detection ofIgAκ and IgAλ in one test sample. This would aid the ultimate challengeof developing a viable multiplex assay system, allowing the simultaneousmeasurement of various monoclonal immunoglobulins, for use in thediagnosis and monitoring of diseases such as multiple myeloma.

REFERENCES

-   Haraldsson A, Kock-Jansen M J, Jaminon M, van Eck-Arts P B, de Boo    T, Weemaes C M, Bakkeren J A. Determination of kappa and lambda    light chains in serum immunoglobulins G, A and M. Ann Clin Biochem    1991; 28: 461-466-   Chui S H, Lam C W, Lewis W H, Lai K N. Light-chain ratio of serum    IgA1 in IgA nephropathy. J Clin Immunol 1991; 11 (4): 219-23

Anti Class—Light Chain Type Antibodies

Polyclonal antibodies against IgGκ were produced using the methodsubstantially as suggested in WO 97/17372. That is, sheep were used toproduce the polyclonal antibody.

Sheep were tolerised at day −3 (that is three days before the mainimmunisation) with 10 mg of IgGλ and IgAκ.

Three days later on day 0, the sheep had the primary immunisationcarried out on it with 50 μg of IgGκ. The IgGλ, IgAκ and IgGκ were humanimmunoglobulins.

Additionally, on day 0, the sheep received anti-human whole λ(100 μg)and sheep anti-human IgA (100 μg). Anti-whole {circle around (2)} bindsboth bound and free {circle around (2)} chains.

On day 42, the sheep had its immunisation boosted with 10 μg of IgGκ.Additionally, it had intravenous administration of sheep anti-humanwhole λ (100 μg) and sheep anti-human IgA (100 μg).

Finally, on day 49, antibody was collected by plasmaphoresis.

The anti-IgGκ was purified by adsorbing with IgMκ and IgGλ. Excessimmune complexes were removed by adding 3% w/v of polyethylene glycol(6,000) and the precipitate was removed by centrifugation.

Positive affinity chromatography was then performed by a passage down anIgGκ column which comprised IgGκ covalently linked to Sepharose 4B. Thebound anti-IgGκ antibodies were then eluted from the column with highsalt buffer.

These antibodies were dialysed against a physiological buffer (phosphatebuffered saline pH7.4 with preservative) and concentrated to 1 g/litre.This concentrated solution was then tested by immunoelectrophoresis(IEP) for specificity. The results are shown in FIG. 16.

The production of antibodies was repeated so that antibodies against thejunction between heavy and light chains of IgG, and then IgA and IgDwere obtained. These were used to identify and quantify intact myelomaimmunoglobulins.

Anti-IgGκ Heavy Chain Antibodies:

For antibodies against IgGκ heavy chains, antisera was usednephalometrically to produce a calibration curve.

Conc (mg/L) 23.6187 47.2375 94.475 188.95 377.9 755.8 Value (Bit) 5191448 3028 5660 8809 12096

The calibration curve shape corresponded to the one expected fromsimilar experiments using Freelite.

Known concentrations of different myeloma samples were then testedagainst the calibration curve, in order to test the efficiency of theantibodies by comparing the sample known concentration to the one givenusing the nephelometric assay.

The results obtained for the determination of IgGκ concentrationscorresponded to those determined by other methods.

Anti-IgGλ Heavy Chains Antibodies:

For antibodies against IgGλ heavy chains, antisera was usednephalometrically to produce a calibration curve.

Conc (mg/L) 23.6187 47.2375 94.475 188.95 377.9 755.8 Value (Bit) 300914 1936 3105 4968 7996

As before, the calibration curve shape corresponded to the one expectedfrom previous experiments involving nephelometric assay.

The quantity of antibodies was not sufficient enough to proceed withfurther tests at that time. However, the calibration curve was similarto the one involving the anti-IgGκ antibodies, with expectation ofsimilar results if tested against known concentration samples.

1. A method of detecting, monitoring or diagnosing a malignant plasmacell disease in a patient, said method comprising obtaining a firstsample from said patient; contacting said sample with at least oneantibody, or a Fab or F(ab′)₂ fragment thereof, specific for the heavychain class; contacting said sample with light chain class antibodies,or a Fab or F(ab′)₂ fragments thereof, said light chain antibodiescomprising antibodies specific for λ light chains and/or antibodiesspecific for κ light chains, wherein one class of antibodies selectedfrom said heavy chain class and light chain antibodies (or theircounterpart Fab or F(ab′)2 fragments thereof), is immobilized on a solidsupport and the other is labeled; removing unbound immunoglobins andfree light chains; conducting an immunoassay on said sample to determinea first ratio between the measured relative amounts of immunoglobulinshaving a heavy chain class bound to λ light chains, compared toimmunoglobulins having the same heavy chain class but bound to κ lightchains; comparing said first ratio to a second ratio, said second ratiocomprising the relative amounts of immunoglobins having a heavy chainclass bound to λ light chains, compared to immunoglobulins having thesame heavy chain class but bound to κ light chains in a) a sampleobtained from an individual without the disease, or b) a second sampletaken from said patient after said first sample; wherein a deviation ofsaid first ratio from said second ratio indicates the presence of amalignant plasma cell disease and/or the progression/regression of thedisease.
 2. The method according to claim 1, wherein the heavy chainclass or Fab or F(ab′)₂ fragment thereof is immobilized on said solidsupport.
 3. The method according to claim 1, wherein the first andsecond ratios are determined using an immunosorbent assay, such as anEnzyme Linked Immunosorbent Assays (ELISA) comprising the antibodies. 4.The method according to claim 1, wherein the heavy chain class antibody,is bound to a bead and flow cytometry is used to determine thekappa/heavy chain to lamda/heavy chain ratios for a given heavy chainclass in said sample.
 5. The method according to claim 1, wherein thebinding of the antibodies to the immunoglobulins in the sample isdetermined by using a nephelometer, a turbidimeter, flow cytometry orcolor coded microspheres.
 6. The method according to claim 1,additionally comprising the step of detecting the presence and/or amountof free λ and/or free κ light chains in the sample.
 7. The methodaccording to claim 6, wherein the free λ and/or free κ light chains aredetected by antibodies specific for free λ or free κ light chains. 8.The method of claim 1 wherein said sample is a blood or serum sample. 9.The method according to claim 1, wherein the heavy chain class, or Fabor F(ab′)₂ fragment thereof, is heavy chain subclass specific.
 10. Amethod of monitoring a malignant plasma cell disease in a patient duringtreatment to determine the efficacy of the treatment, said methodcomprising determining a first ratio between the relative amounts ofimmunoglobulins having: a heavy chain class bound to λ light chains,compared to immunoglobulins having the same heavy chain class but boundto κ light chains in a sample obtained from said patient beforeadministration of said treatment; administering said treatment;determining a second ratio between the relative amounts ofimmunoglobulins having: a heavy chain class bound to λ light chains,compared to immunoglobulins having the same heavy chain class but boundto κ light chains in a sample obtained from said patient afteradministration of said treatment, comparing said first and second ratiosto a threshold value obtained from population data analysis of measuredratios between the amounts of immunoglobulins having: a heavy chainclass bound to λ light chains, compared to immunoglobulins having thesame heavy chain class but bound to κ light chains in samples obtainedfrom individuals without the disease; wherein the efficacy of thetherapy is indicated by the second ratio being more similar to saidthird ratio than the first.
 11. The method of claim 10 wherein saidsample is a blood or serum sample.
 12. The method according to claim 10,wherein the heavy chain class antibody, is bound to a bead and flowcytometry is used to determine the kappa/heavy chain to lamda/heavychain ratios for a given heavy chain class in said sample.
 13. Themethod according to claim 10, wherein the heavy chain class, or Fab orF(ab′)₂ fragment thereof, is heavy chain subclass specific.
 14. A methodof detecting, monitoring or diagnosing a malignant plasma cell diseasein a patient, said method comprising obtaining a sample from saidpatient; contacting said sample with at least one antibody, or a Fab orF(ab′)₂ fragment thereof, specific for the heavy chain class; contactingsaid sample with light chain class antibodies, or a Fab or F(ab′)₂fragments thereof, said light chain antibodies comprising antibodiesspecific for λ light chains and/or antibodies specific for κ lightchains, wherein one class of antibodies selected from said heavy chainclass and light chain antibodies (or their counterpart Fab or F(ab′)2fragments thereof), is immobilized on a solid support and the other islabeled; removing unbound immunoglobins and free light chains;conducting an immunoassay on said sample to determine a first ratiobetween the measured relative amounts of immunoglobulins having a heavychain class bound to λ light chains, compared to immunoglobulins havingthe same heavy chain class but bound to κ light chains; comparing saidfirst ratio to a threshold value obtained from population data analysisof measured ratios between the amounts of immunoglobulins having a heavychain class bound to λ light chains, relative to immunoglobulins havingthe same heavy chain class but bound to κ light chains in samplesobtained from individuals without the disease; wherein a deviation ofsaid first ratio from said threshold value indicates the presence of amalignant plasma cell disease.